Transformant producing secondary metabolite modified with functional group and novel biosynthesis genes

ABSTRACT

An objective of the present invention is to provide a transformant altered so as to produce a secondary metabolite in which a benzene ring of the secondary product is modified at the para-position with a functional group containing a nitrogen atom. A transformant according to the present invention is a transformant of an organism producing a secondary metabolite having a benzene ring skeleton without substitution at the para-position with a functional group containing a nitrogen atom, said transformant being transformed by introducing genes involved in a biosynthetic pathway from chorismic acid to p-aminophenylpyruvic acid, including a gene encoding an amino acid sequence (SEQ ID NO: 2) having 4-amino-4-deoxychorismic acid synthase activity, a gene encoding an amino acid sequence (SEQ ID NO: 4) having 4-amino-4-deoxychorismic acid mutase activity and a gene encoding an amino acid sequence (SEQ ID NO: 6) having 4-amino-4-deoxyprephenic acid dehydrogenase activity, so as to produce a secondary metabolite having a benzene ring skeleton substituted at the para-position with a functional group containing a nitrogen atom. Another objective of the present invention is to provide a novel gene involved in the biosynthetic pathway from chorismic acid to p-aminophenylpyruvic acid. A novel gene according to the present invention comprises genes encoding the amino acid sequences of SEQ ID NOs: 2, 4 and 6 or modified sequences thereof.

This application is a 371 of PCT/JP00/06783 filed Sep. 29, 2000, whichclaims the foreign priority of Japan 11/276314 filed Sep. 29, 1999.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to transformants producing secondarymetabolites modified by functional groups, more specifically, totransformants producing secondary metabolites in which a benzene ring ismodified at the para-position with a functional group containing anitrogen atom. Furthermore, the present invention relates to novel genesinvolved in a biosynthetic pathway from chorismic acid top-aminophenylpyruvic acid.

2. Description of the Related Art

Since organisms produce a number of various kinds of secondarymetabolites having biological activity, research for utilizing thesemetabolites for drugs for humans and animals, agricultural chemicals,and the like has been actively carried out. However, secondarymetabolites from organisms can rarely be utilized for practical use asthey are, and accordingly they are generally modified with variousfunctional groups to optimize their biological activity. A modificationwith a functional group containing a nitrogen atom, such as a nitrogroup and amino group, is one of the most important modifications.

Chemical methods are available for modifying a certain substance with anitro group. However, introduction of a nitro group into a benzene ringspecifically at the para-position using a chemical method is extremelydifficult, and its yield is very low. Furthermore, when a substance tobe modified with a nitro group is as complex as a secondary metabolitefrom an organism, it is even more difficult to specifically modify abenzene ring at the para-position with a nitro group.

On the other hand, methods of introducing an amino group are generallyclassified into two groups, i.e., enzymatic methods and chemicalmethods. In enzymatic methods, an enzyme called aminotransferase (EC2.6.1 group) is used. However, substances which can be a substrate forthe aminotransferase are limited, and no enzyme has been known todirectly transfer an amino group to a benzene ring. Therefore, onlychemical methods have been available for modification of a benzene ringwith an amino group.

However, in chemical procedure, it is necessary to first modify abenzene ring with a nitro group and then to reduce this nitro group intoan amino group. Since the nitration reaction in the first step is verydifficult, introduction of the amino group into the benzene ring bychemical methods is extremely difficult. Accordingly, development of amethod of modifying a benzene ring specifically at the para-positionwith a nitro group or an amino group has been strongly needed.

SUMMARY OF THE INVENTION

An objective of the present invention is to provide a transformantmodified so as to produce a secondary metabolite in which a benzene ringof the secondary product is modified at the para-position with afunctional group containing a nitrogen atom, and a method of producingthe modified secondary metabolite with ease and at low costs.

The present inventors have successfully obtained a transformant thatproduces a substance PF1022 in which a benzene ring is modified at thepara-position with an amino group by transforming a microorganismproducing the substance PF1022 containing a benzene ring skeleton with aDNA containing a gene involved in a biosynthetic pathway from chorismicacid to p-aminophenylpyruvic acid.

A transformant according to the present invention is a transformant ofan organism producing a secondary metabolite having a benzene ringskeleton that is not substituted with a functional group containing anitrogen atom at the para-position, wherein the transformant istransformed by introducing a gene involved in a biosynthetic pathwayfrom chorismic acid to p-aminophenylpyruvic acid (hereinafter refer toas “biosynthesis gene”) so that the transformant produces a secondarymetabolite having a benzene ring skeleton substituted at thepara-position with a functional group containing a nitrogen atom.

A method according to the present invention is a method for producing asecondary metabolite having a benzene ring skeleton substituted at thepara-position with a functional group containing a nitrogen atom, whichcomprises the steps of culturing the above-mentioned transformant andcollecting the secondary metabolite having a benzene ring skeletonsubstituted at the para-position with a functional group containing anitrogen atom.

Another objective of the present invention is to provide a novel geneinvolved in the biosynthetic pathway from chorismic acid top-aminophenylpyruvic acid.

Novel genes according to the present invention are a gene encoding theamino acid sequence of SEQ ID NO: 2 or a modified sequence of SEQ ID NO:2 having 4-amino-4-deoxychorismic acid synthase activity; a geneencoding the amino acid sequence of SEQ ID NO: 4 or a modified sequenceof SEQ ID NO: 4 having 4-amino-4-deoxychorismic acid mutase activity;and a gene encoding the amino acid sequence of SEQ ID NO: 6 or amodified sequence of SEQ ID NO: 6 having 4-amino-4-deoxyprephenic aciddehydrogenase activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the restriction map of a DNA fragment isolated fromStreptomyces venezuelae and the position of open reading frames thereon.

FIG. 2 shows the construction of plasmid pTrc-papA.

FIG. 3 shows the amino acid analyzer chromatograms used for detectingenzyme activity of a papA gene product.

FIG. 4 shows the construction of plasmid pTrc-papB.

FIG. 5 shows the amino acid analyzer chromatograms used for detectingenzyme activity of a papB gene product.

FIG. 6 shows the construction of plasmid pET-papC1.

FIG. 7 shows the amino acid analyzer chromatograms used for detectingenzyme activity of a papC gene product.

FIG. 8 shows the construction of plasmids pPF260-A2 and pPF260-A3.

FIG. 9 shows the restriction map of the 6-kb HindIII fragment containingthe Abp1 gene.

FIG. 10 shows the restriction map of pABPd.

FIG. 11 shows the construction of plasmid pPF260-B3.

FIG. 12 shows the construction of plasmid pPF260-C3.

FIG. 13 shows the HPLC chromatograms used for detecting PF1022derivatives in which a benzene ring is modified at the para-positionwith a nitro group or an amino group.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Deposition of Microorganisms

The strain PF1022 described in Example 5 was deposited with the NationalInstitute of Bioscience and Human-Technology, Agency of IndustrialScience and Technology, the Ministry of International Trade and Industry(1-3 Higashi 1-Chome, Tsukuba City, Ibaraki Prefecture, Japan), datedJan. 24, 1989. The accession number is FERM BP-2671.

The transformant 55-65 of Mycelia sterilia was deposited with theNational Institute of Bioscience and Human-Technology, Agency ofIndustrial Science and Technology, the Ministry of International Tradeand Industry (1-3 Higashi 1-Chome, Tsukuba City, Ibaraki Prefecture,Japan), dated Sep. 17, 1999. The accession number is FERM BP-7255.

Escherichia coli (JM109) transformed with plasmid pUC118-papA wasdeposited with the National Institute of Bioscience andHuman-Technology, Agency of Industrial Science and Technology, theMinistry of International Trade and Industry (1-3 Higashi 1-Chome,Tsukuba City, Ibaraki Prefecture, Japan), dated Sep. 17, 1999. Theaccession number is FERM BP-7256.

Escherichia coli (JM109) transformed with plasmid pTrc-papB wasdeposited with the National Institute of Bioscience andHuman-Technology, Agency of Industrial Science and Technology, theMinistry of International Trade and Industry (1-3 Higashi 1-Chome,Tsukuba City, Ibaraki Prefecture, Japan), dated Sep. 17, 1999. Theaccession number is FERM BP-7257.

Escherichia coli (JM109) transformed with plasmid pET-papC was depositedwith the National Institute of Bioscience and Human-Technology, Agencyof Industrial Science and Technology, the Ministry of InternationalTrade and Industry (1-3 Higashi 1-Chome, Tsukuba City, IbarakiPrefecture, Japan), dated Sep. 17, 1999. The accession number is FERMBP-7258.

Organisms to be Transformed

Organisms to be used in the present invention can be those which producea secondary metabolite containing a benzene ring skeleton. Examples ofpreferable organisms include those which produce a secondary metabolitebiosynthesized via chorismic acid, in particular, a secondary metabolitesynthesized from at least one building block selected from the groupconsisting of phenylpyruvic acid, p-hydroxyphenylpyruvic acid,phenylalanine, tyrosine, and phenyllactic acid.

Examples of preferable organisms also include those which produce apeptide or a depsipeptide, in particular, a peptide or a depsipeptidesynthesized from at least one building block selected from the groupconsisting of phenylalanine, tyrosine, and phenyllactic acid, as asecondary metabolite.

Such secondary metabolites and microorganisms producing the same includethe following.

Names of Compounds and Microorganisms Olstatin D Bacillus cereus KY-21,Nigrosabulum sp. 28Y1 Nannochelin Nannocystis exedens Nae485Phosphonophenylalanylarginine Streptomyces rishiensis NK-122 15B1Actinomadura spiculoosospora K-4 Ahpatinin A Streptomyces sp. WK-142A-38533 Streptomyces sp. Melanostatin Streptomyces clavus N924-1,Streptomyces clavifer N924-2 Aldostatin Pseudoeurotium zonatum M4109N-Acetyl-L-phenylalanyl- Emericellopsis salmosynemata L-phenylalaninolBestatin Streptomyces olivoverticuli Estatin A Myceliophthorathermophilia M4323 N-(N-L-Arginyl-D-allo- Keratinophyton terreum Tu 534threonyl)-L-phenylalanine Streptin P1 Streptomyces tanabeensis WF-10129Doratomyces putredinis F-214690 Clobamide Kobatiella caulivoraSP-Chymostatin B Streptomyces nigrescens WT-27, Streptomyces libani,Streptomyces sp. GE16457 Antipain Streptomyces sp. KC84-AG13 MilolysineK_(A) Metarrhizium anisopliae U-47 Tyrostatin Kitasatospora sp. 55,Streptomyces sp. SAM-0986 Detoxin Streptomyces caespitosus var.detoxicus 7072 GC_(1,) Streptomyces mobaraensis Chymostatin Streptomycessp. Tridecaptin Bacillus polymyxa Alamecycine Trichoderma viridisTrichocerine Trichoderma viride Trichosporin B Trichoderma polysporumTrichodyanine Trichoderma polysporum, Trichoderma harzianum SamarosporinI Emericellopsis microspora, Samarospora sp., Stibella sp. SuzukacylineA Trichoderma viride Trichologin Trichoderma longibrachiatum ZervamicinEmericellopsis microspora, Emericellopsis salmosynnemata AntianiebinEmericellopsis synnematicola, Emericellopsis poonesis, Cephalosporumpimprina Gramicidin C Bacillus brevis Ochratoxins Aspergillus ochraceus,Aspergillus melleus, Aspergillus sulphureus, Penicillium viridicatumFR-900261 Petriella sp., Petriella guttulata 3161 ChiamydocineDiheterospora chlamydosporia Trapoxin Helicoma ambiens RF-1023 Cyl-1Cylindrocladium scoparium Cyl-2 Cylindrocladium scoparium AspercholineAspergillus versicolor Lotusine Zizyphus lotus Lyciumin Lycium chinenseMill. Avellanin Hamigera avellanea, Penicillium sp. PF1119 CycloasptideAspergillus sp. NE-45 Bouvardine Bouvardia ternifolia, Rubia cordifoliaCycloamanide A Amanitia phalloides Cycloamanide B Amanitia phalloidesHeterophyllin A Pseudostellarea heterophylla Polymyxin Bacillus polymyxaOctapeptin Bacillus cirrculans G493-B6, Bacillus sp. JP301 Bu-2470ABacillus circulans Mucosubtilin Bacillus subtillis Bacillomycin DBacillus subtilis I-164, Bacillus subtilis Sc-3 Iturin A Bacillussubtilis Cyanogicin Microcystis aeruginosa Bacitracin Bacillus subtilis,Bacillus licheniformis Gramicidin S Bacillus brevis Antamanide Amanitiaphalloides Tyrocidin Bacillus brevis Cortinarine Cortinariusspeciosissimus Mycobacillin Bacillus subtilis TL119 Bacillus subtilisBeauverolide Beauveria bassiana, Isaria sp. NeoantimycinStreptoverticillium orinoci MK3990 Basidiobolus sp. MK3990 LeualacinHapsidospora irregularis SANK 17182 A54556 Streptomyces hawaiiensisEnopeptin B Streptomyces sp. RK-1051 Beauvaricin Beauveria bassianaXanthostatin Streptomyces spiroverticillatus Valiapeptin Streptomycescitrus K3619, Streptomyces flavidovirens Verginiamycin S₁ Streptomycesvirginiae, Streptomyces Alborectus Cycloheptamycin Streptomyces sp.WS-9326 Streptomyces violaceusniger 9326 Fusaria fungi Fusariumsporotrichoides, Fusarium cyclodepsipeptide roseum, Fusarium tricinctumFR-900359 Ardisia crenata Verlamellin Verticillium lamellicola MF4683Didemnin Trididermnum solidam Lipopeptin A Streptomyces sp. AC-69 20561Aeromonas sp. Neopeptin Streptomyces sp. K-710 AureobasidinAureobasidium pullulans R106 Syringomycin Pseudomonas syringae pv.Syringae Plipastatin Bacillus cereus BMG302-fF67 Permetin A Bacilluscirculans H913-B4 BMY-28160 Bacillus circulans Polypeptin A Bacilluscirculans Brevistin Bacillus brevis Ramoplanin Actinoplanes sp.ATCC33076 Ancovenin Streptomyces sp. A-647P Duramycins Streptomyceshachijoensis var. takahagiensis E-312 Streptoverticillium griseoluteus2075, Streptoverticillium griseoverticillatum PA-48009 CinnamycinStreptomyces cinnamoneus Actinoidin Nocardia actinoides SKF-AAJ-193,Proactinomycetes actinoides Substance PF1022 Mycelia sterilia

These substances are described in the Dictionary of Natural Products(Chapman & Hall, 1994).

“Organisms” to be transformed in the present invention includemicroorganisms such as bacteria, yeasts and fungi, and plants. Theplants also include plant cells.

Examples of the functional group containing a nitrogen atom include anamino group and nitro group.

When an organism to be transformed is a microorganism that produces thesubstance PF1022 represented by the following formula:

the secondary metabolite produced by the transformant can be thesubstance PF1022 represented by the following formula, which is modifiedwith an amino group (hereinafter refer to as “substance PF1022derivative”).

The substance PF1022[cyclo(D-lactyl-L-N-methylleucyl-D-3-phenyllactyl-L-N-methylleucyl-D-lactyl-L-N-methylleucyl-D-3-phenyllactyl-L-N-methylleucyl)]is a cyclic depsipeptide that is produced by the filamentous fungusstrain PF1022 (Mycelia sterilia, FERM BP-2671), which belongs toAgonomycetales and has extremely high anthelmintic activity againstanimal parasitic nematodes (Japanese Patent Application Laid-openPublication No. 35796/1991; Sasaki, T. et al., J. Antibiotics, 45, 692,1992). Accordingly, the substance PF1022 is useful as an anthelmintic,and also a derivative thereof modified with an amino group is useful asa raw material for synthesizing a highly active derivative of thissubstance.

The substance PF1022 is synthesized by a substance PF1022-synthesizingenzyme from four molecules of L-leucine, two molecules of D-lactic acid,and two molecules of D-phenyllactic acid. Not restricted to thefollowing, it is thought that (1) p-aminophenylpyruvic acid is producedin a transformant by introducing a gene involved in the biosyntheticpathway from chorismic acid to p-aminophenylpyruvic acid into asubstance PF1022-producing microorganism, (2) D-phenyllactic aciddehydrogenase (D-PLDH) acts on the resulting product to producep-amino-D-phenyllactic acid in the transformant, (3) the substancePF1022-synthesizing enzyme acts on p-amino-D-phenyllactic acid insteadof D-phenyllactic acid, and thus (4) the substance PF1022 derivative isproduced.

Biosynthesis Genes

Examples of enzymes involved in the biosynthesis from chorismic acid top-aminophenylpyruvic acid include 4-amino-4-deoxychorismic acidsynthase, 4-amino-4-deoxychorismic acid mutase, and4-amino-4-deoxyprephenic acid dehydrogenase (Blanc, V. et al., Mol.Microbiol., 23, 191–202, 1997). The biosynthetic pathway from chorismicacid to p-aminophenylpyruvic acid can be summarized as follows:4-Amino-4-deoxychorismic acid synthase acts on chorismic acid to produce4-amino-4-deoxychorismic acid; 4-amino-4-deoxychorismic acid mutase actson the resulting 4-amino-4-deoxychorismic acid to produce4-amino-4-deoxyprephenic acid; and 4-amino-4-deoxyprephenic aciddehydrogenase acts on the resulting 4-amino-4-deoxyprephenic acid toproduce p-aminophenylpyruvic acid.

The 4-amino-4-deoxychorismic acid synthase includes an enzyme that actson chorismic acid to transform it into 4-amino-4-deoxychorismic acid.

The 4-amino-4-deoxychorismic acid synthase is found in a wide variety oforganisms as a part of the biosynthesis system from chorismic acid top-aminobenzoic acid. p-Aminobenzoic acid is synthesized from chorismicacid in a two-step reaction. The former reaction is catalyzed by4-amino-4-deoxychorismic acid synthase, and the latter reaction iscatalyzed by 4-amino-4-deoxychorismic acid lyase (Green, J. M. andNichols, B. P., J. Biol. Chem., 266, 12971–12975, 1991).

Reported genes encoding 4-amino-4-deoxychorismic acid synthase includethose derived from Escherichia coli (Kaplan, J. B. and Nichols, B. P.,J. Mol. Biol., 168, 451–468, 1983); Goncharoff, P. and Nichols, B. P.,J. Bacteriol., 159, 57–62, 1984), Bacillus subtilis(Slock, J. et al., J.Bacteriol., 172, 7211–7226, 1990), Klebsiella pneumoniae (Kaplan, J. B.et al., J. Mol. Biol., 183, 327–340, 1985; Goncharoff, P. and Nichols,B. P., Mol. Biol. Evol., 5, 531–548, 1988), Streptomycespristinaespiralis (Blanc, V. et al., Mol. Microbiol., 23, 191–202,1997), Streptomyces venezuelae (Brown, M. P. et al., Microbiology, 142,1345–1355, 1996), and Saccharomyces cerevisiae (Edman, J. C. et al.,Yeast, 9, 669–675, 1993), and they can be used. Genes encoding the4-amino-4-deoxychorismic acid synthase, other than those mentionedabove, can also be isolated from organisms having4-amino-4-deoxychorismic acid synthase activity using standardtechniques and used in the present invention.

On the other hand, the 4-amino-4-deoxychorismic acid synthase can begenerally divided into two groups: one which is composed of twopolypeptides, such as those derived from Escherichia coli, Bacillussubtilis, or Klebsiella pneumoniae, and the other which is composed ofone peptide, such as those from a part of Actinomycetes or Saccharomycescerevisiae. In the present invention, it is preferable to use a geneencoding the 4-amino-4-deoxychorismic acid synthase consisting of onepolypeptide since a plurality of genes has to be introduced to a host.

In the present invention, an example of the gene encoding the4-amino-4-deoxychorismic acid synthase is preferably a gene encoding theamino acid sequence of SEQ ID NO: 2 or a modified sequence of SEQ IDNO:2 having 4-amino-4-deoxychorismic acid synthase activity, morepreferably a gene containing the DNA sequence of SEQ ID NO: 1.

In the present invention, “modified sequence” means a sequence havingone or more, for example one to several, modifications selected from thegroup consisting of a substitution, a deletion, an insertion, and anaddition.

In the present invention, whether a modified amino acid sequence “has4-amino-4-deoxychorismic acid synthase activity” or not can be evaluatedby allowing the protein comprising said amino acid sequence to act on asubstrate and then detecting the reaction product, for example,according to the method described in Example 2.

The 4-amino-4-deoxychorismic acid mutase means an enzyme that acts on4-amino-4-deoxychorismic acid to transform it into4-amino-4-deoxyprephenic acid.

The 4-amino-4-deoxyprephenic acid dehydrogenase means an enzyme whichacts on 4-amino-4-deoxyprephenic acid to transform it intop-aminophenylpyruvic acid.

A gene encoding 4-amino-4-deoxychorismic acid mutase and a gene encoding4-amino-4-deoxyprephenic acid dehydrogenase are obtained from organismsthat can biosynthesize p-aminophenylpyruvic acid. More specifically,examples of such organisms include Streptomyces pristinaespiralis thatproduces pristinamycin I; Streptomyces loidensis that producesvernamycin B; Corynebacterium hydrocarboclastus that produce corynecin;and Streptomyces venezuelae that produces chloramphenicol. Among theseorganisms, Streptomyces pristinaespiralis can be used in the presentinvention since genes which presumably encode 4-amino-4-deoxychorismicacid mutase and 4-amino-4-deoxyprephenic acid dehydrogenase have alreadybeen isolated and their nucleotide sequences have been determined (V.Blanc et al., Mol. Microbiol., 23, 191–202, 1997).

A number of genes encoding chorismic acid mutase and prephenic aciddehydrogenase have been already isolated from bacteria, yeasts, plantsand the like, and these genes can be modified by substituting, deletingor adding appropriate amino acids so as to have 4-amino-4-deoxychorismicacid mutase activity and 4-amino-4-deoxyprephenic acid dehydrogenaseactivity, based on protein engineering techniques or directed evolutiontechniques. Thus, the resulting modified genes can also be used in thepresent invention.

In the present invention, an example of the gene encoding the4-amino-4-deoxychorismic acid mutase is preferably a gene encoding theamino acid sequence of SEQ ID NO: 4 or a modified sequence of SEQ IDNO:4 having 4-amino-4-deoxychorismic acid mutase activity, morepreferably a gene containing the DNA sequence of SEQ ID NO: 3.

In the present invention, whether a modified amino acid sequence “has4-amino-4-deoxychorismic acid mutase activity” or not can be evaluatedby allowing the protein comprising said amino acid sequence to act on asubstrate and then detecting the reaction product, for example,according to the method described in Example 3.

In the present invention, an example of the gene encoding the4-amino-4-deoxyprephenic acid dehydrogenase is preferably a geneencoding the amino acid sequence of SEQ ID NO: 6 or a modified sequenceof SEQ ID NO: 6 having 4-amino-4-deoxyprephenic acid dehydrogenaseactivity, more preferably a gene containing the DNA sequence of SEQ IDNO: 5.

In the present invention, whether a modified amino acid sequence “has4-amino-4-deoxyprephenic acid dehydrogenase activity” or not can beevaluated by allowing the protein comprising said amino acid sequence toact on a substrate and then detecting the reaction product, for example,according to the method described in Example 4.

Given the amino acid sequences of enzymes involved in the biosynthesisin the present invention, nucleotide sequences encoding the amino acidsequences can be easily determined, and various nucleotide sequencesencoding the amino acid sequences depicted in SEQ ID NOs: 2, 4, and 6can be selected. Accordingly, biosynthesis genes according to thepresent invention include, in addition to a part or all of the DNAsequences of SEQ ID NOs: 1, 3, and 5, DNA sequences encoding the sameamino acid sequences and having degenerate codons, and further includeRNA sequences corresponding to these sequences.

Transformants

A transformant of the present invention can be obtained by introducing aDNA molecule, in particular an expression vector, comprising a geneinvolved in the biosynthesis from chorismic acid to p-aminophenylpyruvicacid into a host, wherein the DNA molecule is replicable and the genecan be expressed.

In the present invention, when a plurality of biosynthesis enzyme genesis introduced into the host, each gene can be contained in either thesame or different DNA molecules. Further, when the host is a bacterium,each gene can be designed to be expressed as a polycistronic mRNA so asto be made into a single DNA molecule.

The expression vector to be used in the present invention can beappropriately selected from viruses, plasmids, cosmid vectors, and thelike taking the kind of the host cell to be used into consideration. Forexample, lambda bacteriophages and pBR and pUC plasmids can be used whenthe host cell is Escherichia coli; pUB plasmids can be used for Bacillussubtilis; and YEp, YRp, YCp, and YIp plasmid vectors can be used foryeasts.

Among the plasmid vectors to be used, at least one vector preferablycontains a selectable marker to select transformants. A drug resistancegene or a gene complementing an auxotrophic mutation can be used as aselectable maker. Preferable examples of the marker genes to be used foreach host include an ampicillin resistance gene, a kanamycin resistancegene and a tetracycline gene for bacteria; a tryptophan biosynthesisgene (TRP1), an uracil biosynthesis gene (URA3) and a leucinebiosynthesis gene (LEU2) for yeasts; a hygromycin resistance gene, abialaphos resistance gene, a bleomycin resistance gene and anaureobasidin resistance gene for fungi; and a kanamycin resistance geneand a bialaphos resistance gene for plants.

Furthermore, in an expression vector, regulatory sequences necessary forexpression of the individual genes, for example, transcriptionregulatory signals and translation regulatory signals, such as apromoter, a transcription initiation signal, a ribosome binding site, atranslation stop signal, and a transcription stop signal, can operablybe linked to the biosynthesis gene. The regulatory sequences can beselected and ligated according to an ordinary method.

For example, promoters such a lactose operon and a tryptophan operon canbe used in Escherichia coli; promoters of an alcohol dehydrogenase gene,an acid phosphatase gene, a galactose utilization gene, and aglyceraldehyde 3-phosphate dehydrogenase gene can be used in yeasts;promoters such as α-amylase gene, a glucoamylase gene, acellobiohydrolase gene, a glyceraldehyde 3-phosphate dehydrogenase gene,and an Abp1 gene can be used in fungi; and the CaMV 35SRNA promoter, aCaMV 19SRNA promoter, a noparin synthase gene promoter can be used inplants.

Transformation of an organism can be carried out according to anordinary method such as the calcium ion method, the lithium ion method,the electroporation method, the PEG method, the Agrobacterium method,and the particle gun method, and the method can be selected depending onthe organism to be transformed.

In the present invention, a transformant is cultured, and the resultantculture is used to obtain a modified secondary metabolite of interest.The transformant can be cultured also according to an ordinary method byappropriately selecting a medium, culture conditions, and the like.

The medium can be supplemented with a carbon source and nitrogen sourcethat can be anabolized and utilized, respectively, by the transformantof the present invention, various vitamins, various amino acids such asglutamic acid and asparagine, trace nutrients such as nucleotides, andselective agents such as antibiotics. Further, organic and inorganicsubstances that help the growth of the transformant of the presentinvention or promote the production of the secondary metabolite ofinterest can be appropriately added. Further, if necessary, a syntheticmedium or complex medium which appropriately contains other nutrientscan be used.

Any kind of carbon source and nitrogen source can be used in the mediumas long as they can be utilized by the transformant of the presentinvention. As the anabolizable carbon source, for example, variouscarbohydrates, such as sucrose, glucose, starch, sucrose, glycerol,fructose, maltose, mannitol, xylose, galactose, ribose, dextrin, animaland plant oils and the like, or hydrolysates thereof, can be used. Thepreferable concentration generally is from 0.1% to 5% of the medium.

As the utilizable nitrogen source, for example, animal or plantcomponents, or exudates or extracts thereof, such as peptone, meatextract, corn steep liquor, and defatted soybean powder, organic acidammonium salts such as succinic acid ammonium salts and tartaric acidammonium salts, urea, and other various inorganic or organicnitrogen-containing compounds can be used.

Further, as inorganic salts, for example, those which can producesodium, potassium, calcium, magnesium, cobalt, chlorine, phosphoricacid, sulfuric acid, and other ions can be appropriately used.

Of course, any medium which contains other components, such as cells,exudates or extracts of microorganisms such as yeasts, and fine plantpowders, can be appropriately used as long as they don't interfere withthe growth of the transformant and the production and accumulation ofthe secondary metabolite of interest. When a mutant strain having anutritional requirement is cultured, a substance to satisfy itsnutritional requirement is added to the medium. However, this kind ofnutrient may not necessarily be added when a medium containing naturalsubstances is used.

The pH of the medium is, for example, about 6 to 8. Incubation can becarried out by a shaking culture method under an aerobic condition, anagitation culture method with aeration, or an aerobic submerged culturemethod. An appropriate incubation temperature is 15° C. to 40° C.,generally about 26° C. to 37° C. Production of the secondary metaboliteof interest depends on a medium, culture conditions, or a host used.However, the maximum accumulation can generally be attained in 2 to 25days by any culture method. The incubation is terminated when the amountof the secondary metabolite of interest in the medium reaches its peak,and the target substance is isolated from the culture and then purified.

Needless to say, the culture conditions such as the medium component,medium fluidity, incubation temperature, agitation speed and aerationrate can be appropriately selected and controlled depending on thetransformant to be used and the exterior conditions so as to obtainpreferable results. If foaming occurs in a liquid medium, a defoamingagent such as silicone oil, vegetable oils, mineral oils, andsurfactants can be appropriately used. The secondary metabolite ofinterest accumulated in the culture thus obtained is contained in thecells of the transformant of the present invention and the culturefiltrate. Accordingly, it is possible to recover the secondarymetabolite of interest from both culture filtrate and transformant cellsby separating the culture into each fraction by centrifugation.

The secondary metabolite of interest can be recovered from the culturefiltrate according to an ordinary method. Procedures for recovering thesecondary metabolite of interest from the culture can be carried outsingly, in combination in a certain order, or repeatedly. For example,extraction filtration, centrifugation, salting out, concentration,drying, freezing, adsorption, detaching, means for separation based onthe difference in solubility in various solvents, such as precipitation,crystallization, recrystallization, reverse solution, counter-currentdistribution, and chromatography can be used.

Further, the secondary metabolite of interest can be obtained from theculture inside the cells of the transformant of the present invention.For example, extraction from the culture (e.g., smashing and pressuredisruption), recovery (e.g., filtration and centrifugation), andpurification (e.g., salting out and solvent precipitation) can becarried out using an ordinary method.

The crude substance obtained can be purified according to an ordinarymethod, for example, by column chromatography using a carrier such assilica gel and alumina or reverse-phase chromatography using an ODScarrier. A pure secondary metabolite of interest can be obtained fromthe culture of the transformant of the present invention using theabove-mentioned methods, either singly or in appropriate combination.

Transformants Producing Substance PF1022 Derivative

A preferable embodiment of the present invention provides a transformantof a substance PF1022-producing microorganism into which a gene involvedin the biosynthetic pathway from chorismic acid to p-aminophenylpyruvicacid (biosynthesis gene) is introduced.

This transformant can produce a substance PF1022 derivative.

The substance PF1022-producing microorganism to be transformed can beMycelia sterilia, preferably the strain deposited with the NationalInstitute of Bioscience and Human-Technology under an accession numberof FERM BP-2671.

The biosynthesis gene can comprise a gene encoding4-amino-4-deoxychorismic acid synthase, a gene encoding4-amino-4-deoxychorismic acid mutase, and a gene encoding4-amino-4-deoxyprephenic acid dehydrogenase. The gene encoding4-amino-4-deoxychorismic acid synthase can preferably be a gene encodingthe amino acid sequence of SEQ ID NO: 2 or a modified sequence of SEQ IDNO: 2 having 4-amino-4-deoxychorismic acid synthase activity. The geneencoding 4-amino-4-deoxychorismic acid mutase can preferably be a geneencoding the amino acid sequence of SEQ ID NO: 4 or a modified sequenceof SEQ ID NO: 4 having 4-amino-4-deoxychorismic acid mutase activity.The gene encoding 4-amino-4-deoxyprephenic acid dehydrogenase canpreferably be a gene encoding the amino acid sequence of SEQ ID NO: 6 ora modified sequence of SEQ ID NO: 6 having 4-amino-4-deoxyprephenic aciddehydrogenase activity.

An expression vector to be used for transformation is preferably anexpression vector in which the biosynthesis gene is operably linked to aregulatory sequence (e.g., promoter, terminator) which functions in asubstance PF1022-producing microorganism, most preferably an expressionvector in which the biosynthesis gene is operably linked to a regulatorysequence which functions in the strain PF1022 (Mycelia sterilia, FERMBP-2671).

The transformant can be the transformant 55-65 strain deposited with theNational Institute of Bioscience and Human-Technology under an accessionnumber of FERM BP-7255.

Another embodiment of the present invention provides a method ofproducing a substance PF1022 derivative, which comprises the steps ofculturing a transformant of a substance PF1022-producing microorganismand recovering the substance PF1022 derivative from the culture.

EXAMPLE

The present invention is further illustrated by the following examplesthat are not intended as a limitation.

Example 1 Isolation of a Gene Encoding 4-amino-4-deoxychorismic AcidSynthase, a Gene Encoding 4-amino-4-deoxychorismic Acid Mutase, and aGene Encoding 4-amino-4-deoxyprephenic Acid Dehydrogenase fromStreptomyces venezuelae

(1) Preparation of Probe DNA Fragment

A 50 ml portion of a liquid medium (2% soluble starch, 1% polypeptone,0.3% meat extract, 0.05% potassium dihydrogenphosphate, pH 7.0) wasprepared in a 250-ml Erlenmeyer flask. The ISP5230 strain and 140-5strain of Streptomyces venezuelae were each inoculated into this mediumand cultured at 28° C. for 24 hours. After culturing, the cells wereharvested from the culture by centrifugation, and the chromosome DNA wasprepared from these cells by the method described in GeneticManipulation of Streptomyces, A Laboratory Manual (D. A. Hopwood et al.,The John Innes Foundation, 1985).

Next, PCR was carried out using the above-mentioned chromosomal DNA ofthe Streptmyces venezuelae strain ISP5230 as a template andoligonucleotides of SEQ ID NO: 7 and SEQ ID NO: 8 as primers. The PCRwas carried out with a TaKaRa LA PCR™ kit Ver. 2.1 (Takara Shuzo Co.,Ltd.) and Gene Amp PCR System 2400 (Perkin-Elmer). A reaction solutioncontaining 1 μl of the chromosomal DNA (equivalent to 0.62 μg), 5 μl of10-fold concentrated reaction buffer attached to the kit, 8 μl of a 2.5mM dNTP solution, 0.5 μl each of the above-mentioned primers prepared ata concentration of 100 pmol/μl, 5 μl of dimethyl sulfoxide (Wako PureChemical Industries, Ltd.), 0.5 μl of TaKaRa LA-Taq (2.5 U), and 29.5 μlof sterile water was made up into a total volume of 50 μl. The reactionwas carried out by repeating incubation of 25 cycles of one minute at94° C., one minute at 50° C. and 3 minutes at 72° C., after pretreatmentat 94° C. for 10 minutes. After the reaction, a portion of the reactionsolution was subjected to agarose gel electrophoresis to confirm that aDNA fragment of approximately 2 kbp was specifically amplified. Then,the remaining reaction solution was extracted withphenol:chloroform:isoamyl alcohol (25:24:1) and precipitated withethanol. The precipitate was redissolved in sterile water, and theresulting solution (60 μl) was digested with restriction enzyme BamHI,after which agarose gel electrophoresis was carried out, and a band ofapproximately 2 kbp was isolated according to an ordinary method torecover a DNA fragment.

This DNA fragment was cloned into the BamHI site of plasmid pTrcHis B(Invitrogen). Since the restriction map of the inserted fragment of theresulting plasmid was identical to that of pabAB gene (U21728) reportedby Brown et al. (M. P. Brown et al., Microbiology, 142, 1345–1355,1996), the pabAB gene was considered to be cloned, and the plasmid wasnamed pTH-PAB. The plasmid pTH-PAB was digested with restriction enzymeBamHI, agarose gel electrophoresis was carried out, and an insertionfragment was isolated and recovered to be used as a probe for thescreening of a chromosomal DNA library described below.

(2) Screening of Chromosomal DNA Library and Isolation of Genes

About 10 μl of the chromosomal DNA of the Streptomyces venezuelae 140-5strain was partly digested with restriction enzyme Sau3AI, after whichagarose gel electrophoresis was carried out to isolate and recover DNAfragments of from 10 kbp to 20 kbp.

About 0.5 μg of the DNA fragments of from 10 kbp to 20 kbp thusrecovered and 1 μl of λDASH II previously double-digested withrestriction enzymes BamHI and XhoI were ligated with T4 DNA ligase andthen packaged in vitro using a Gigapack III packaging extract(Stratagene) to construct a chromosomal DNA library. Plaques were formedby infecting Escherichia coli XLI-Blue MRA with this DNA library.

Plaque hybridization was carried out using the DNA fragment ofapproximately 2 kbp isolated in (1) as a probe and an ECL Direct DNA/RNALabeling Detection System (Amersham Pharmacia Biotech) to screen about24000 plaques. Among positive clones thus obtained, ten clones weresubjected to a secondary screening, and the resulting positive cloneswere purified to prepare phage DNAs.

These phage DNAs were digested with restriction enzyme BamHI, andSouthern analysis was carried out, which revealed that the probe washybridized with two kinds of DNA fragments, i.e., fragments ofapproximately 1.8 kbp and approximately 3.4 kbp. Further, restrictionmap analysis of the phage DNAs revealed that these two kinds of DNAfragments were adjoining on the chromosomal DNA.

Next, the entire nucleotide sequences of these two kinds of DNAfragments were determined using a fluorescent DNA sequencer ABI PRISM377 (Perkin-Elmer). As a result of the subsequent open-reading-frame(ORF) search, ORFs I–IV were found as shown in FIG. 1. The amino acidsequences deduced from each of the ORFs were searched for homology withknown amino acid sequences using database, which revealed that ORF I washomologous to p-aminobenzoic acid-synthesizing enzyme, ORF II washomologous to prephenic acid dehydrogenase, and ORF III was homologousto chorismic acid mutase. Genes of ORF I, II and III were then namedpapA, papCand papB, respectively. The amino acid sequence encoded bypapA and the nucleotide sequence of papA are each shown in SEQ ID NO: 2and SEQ ID NO: 1; the amino acid sequence encoded by papB and thenucleotide sequence of papB are each shown in SEQ ID NO: 4 and SEQ IDNO: 3; and the amino acid sequence encoded by papC and the nucleotidesequence of papC are each shown in SEQ ID NO: 6 and SEQ ID NO: 5.

Example 2 Expression of papA Gene in Escherichia coli

In order to obtain the translation region of the papA gene, PCR wascarried out with the phage DNA derived from the positive clone shown inExample 1 as a template and oligonucleotides of SEQ ID NO: 9 and SEQ IDNO: 10 as primers. The PCR was carried out with KOD Dash (Toyobo Co.,Ltd.) as DNA polymerase using the Gene Amp PCR System 9700(Perkin-Elmer). A reaction solution containing 1 μl of phage DNA(equivalent to 1 μg), 5 μl of 10-fold concentrated reaction bufferattached to the enzyme, 5 μl of a 2 mM dNTP solution, 1 μl each of theabove-mentioned primers prepared at a concentration of 100 pmol/μl, 5 μlof dimethyl sulfoxide (Wako Pure Chemical Industries, Ltd.), 1 μl of KODDash, and 31 μl of sterile water was made up into a total volume of 50μl. The reaction was carried out by repeating incubation of 15 cycles of30 seconds at 94° C., 2 seconds at 50° C. and 30 seconds at 72° C.,after pretreatment at 94° C. for 5 minutes. The reaction solution thusobtained was extracted with phenol:chloroform:isoamyl alcohol (25:24:1)and precipitated with ethanol. The precipitate was redissolved insterile water, and the DNA terminals were blunted using a DNA bluntingkit (Takara Shuzo Co., Ltd.). Further, the 5′ end was phosphorylatedusing T4 DNA kinase (Wako Pure Chemical Industries, Ltd.), after whichagarose gel electrophoresis was carried out, a DNA fragment ofapproximately 2 kbp was isolated, recovered, and cloned into the SmaIsite of plasmid pUC118 to obtain plasmid pUC118-papA.

The nucleotide sequence of the inserted fragment of pUC118-papA wasdetermined using a fluorescent DNA sequencer ABI PRISM 310 GeneticAnalyzer (Perkin-Elmer). As a result, it was revealed that cytosine atposition 2043 in the nucleotide sequence of SEQ ID NO: 1 was replaced byadenine. Since this replacement was believed to be an error uponamplification of the DNA fragment by PCR and brought no change in theamino acid sequence to be encoded, the inserted fragment of pUC118-papAwas used for the following experiment.

pUC118-papA was introduced into Escherichia coli JM110, and a plasmidwas prepared from the resultant transformant using an ordinary method.After digesting with restriction enzyme BclI, agarose gelelectrophoresis was carried out to isolate and recover a BclI DNAfragment of approximately 2 kbp.

On the other hand, plasmid pTrc99A (Amersham Pharmacia Biotech) wasdigested with restriction enzyme NcoI, and the DNA terminals wereblunted using Mung Bean Nuclease (Wako Pure Chemical Industries, Ltd.).The resultant fragment was further digested with restriction enzyme SmaIand then self-ligated using T4 DNA ligase to obtain plasmid pTrc101.

pTrc101 was digested with restriction enzyme BamHI and treated withalkaline phosphatase (Takara Shuzo Co., Ltd.), after which the resultantfragment was ligated to the above-mentioned 2 kbp BclI DNA fragment. Aplasmid into which the papA gene was inserted in the correct orientationto the promoter contained in pTrc101 was selected and named pTrc-papA.FIG. 2 shows the process of the above-mentioned plasmid construction.

The Escherichia coli JM109 strain carrying pTrc-papA was cultured in anLB liquid medium (1% Bacto-tryptone, 0.5% yeast extract, 0.5% sodiumchloride) supplemented with 100 μg/ml ampicillin, at 37° C. overnight. A1 ml portion of the resultant culture was inoculated into 100 ml of thesame medium, and incubation was carried out at 30° C. for 4 hours, afterwhich 1 ml of 100 mM isopropylthiogalactoside (IPTG) was added, andincubation was further carried out at 30° C. for 3 hours. Afterincubation, cells were recovered from the culture by centrifugation,suspended in 4 ml of buffer solution for cell homogenization (50 mMTris-HCl (pH 8.0), 5 mM EDTA, 10% glycerol) and then homogenized byultrasonic treatment. After homogenization, the supernatant wasrecovered by centrifugation to obtain a cell extract. Further, theEscherichia coli JM109 strain carrying plasmid pTrc101 was treated inthe same manner to prepare another cell extract.

The cell extracts thus prepared were measured for their enzymaticactivity. Namely, 100 μl of the cell extract, 400 μl of distilled water,and 500 μl of a substrate solution [10 mM barium chorismate (Sigma), 10mM glutamine (Wako Pure Chemical Industries, Ltd.), 10 mM magnesiumchloride, 100 mM MOPS (Wako Pure Chemical Industries, Ltd.), pH 7.5]were mixed and reacted at 30° C. for 2 hours. After reaction, a portionof the reaction solution was analyzed using a full automatic amino acidanalyzer JLC-500/V (JEOL, Ltd.).

As shown in FIG. 3, when the cell extract prepared from the Escherichiacoli carrying pTrc-papA was used, a peak was detected on a positionshowing the same retention time with a standard for4-amino-4-deoxychorismic acid synthesized according to the method ofChia-Yu P. Teng et al. (Chia-Yu P. Teng et al., J. Am. Chem. Soc., 107,5008–5009, 1985). On the other hand, the peak on that position was notfound when the cell extract was boiled or when the cell extract preparedfrom the Escherichia coli carrying pTrc101 was used. Thus, the papA genewas verified to encode 4-amino-4-deoxychorismic acid synthase.

Example 3 Expression of papB Gene in Escherichia coli

In order to obtain the translation region of the papA gene, PCR wascarried out with the phage DNA derived from the positive clone shown inExample 1 as a template and oligonucleotides of SEQ ID NO: 11 and SEQ IDNO: 12 as primers. The PCR was carried out with KOD Dash (Toyobo Co.,Ltd.) as DNA polymerase using Gene Amp PCR System 9700 (Perkin-Elmer). Areaction solution containing 1 μl of phage DNA (equivalent to 1 μg), 5μl of 10-fold concentrated reaction buffer attached to the enzyme, 5 μlof a 2 mM dNTP solution, 1 μl each of the above-mentioned primersprepared at a concentration of 100 pmol/l, 5 μl of dimethyl sulfoxide(Wako Pure Chemical Industries, Ltd.), 1 μl of KOD Dash and 31 μl ofsterile water was made up into a total volume of 50 μl. The reaction wascarried out by repeating incubation of 15 cycles of 30 seconds at 94°C., 2 seconds at 50° C. and 30 seconds at 72° C., after pretreatment at94° C. for 5 minutes. The reaction solution thus obtained was extractedwith phenol:chloroform:isoamyl alcohol (25:24:1) and precipitated withethanol. The precipitate was redissolved in sterile water and digestedwith restriction enzyme BamHI, after which agarose gel electrophoresiswas carried out, and a DNA fragment of approximately 0.3 kbp wasisolated according to an ordinary method to recover a DNA fragment.

pTrc101 was digested with restriction enzyme BamHI and treated withalkaline phosphatase (Takara Shuzo Co., Ltd.), after which the resultantfragment was ligated to the above-mentioned 0.3-kbp BamHI DNA fragmentusing T4 DNA ligase. A plasmid into which the papB gene was inserted inthe correct orientation to the promoter contained in pTrc101 wasselected and named pTrc-papB (FIG. 4). The nucleotide sequence of theinserted fragment of pTrc-papB was determined using a fluorescent DNAsequencer ABI PRISM 310 Genetic Analyzer (Perkin-Elmer) to verify thatthe sequence was identical with the nucleotide sequence of SEQ ID NO: 3.

The Escherichia coli JM109 strain carrying pTrc-papB was cultured in anLB liquid medium (1% Bacto-tryptone, 0.5% yeast extract, 0.5% sodiumchloride) supplemented with 100 μg/ml ampicillin, at 37° C. overnight. A1 ml portion of the resultant culture was inoculated into 100 ml of thesame medium, and incubation was carried out at 37° C. for 2 hours, afterwhich 1 ml of 100 mM isopropylthiogalactoside (IPTG) was added, andincubation was further carried out at 37° C. for 5 hours. Afterincubation, cells were recovered from the culture by centrifugation,suspended in 4 ml of buffer solution for cell homogenization (50mMTris-HCl (pH 8.0), 5 mM EDTA, 10% glycerol), and then homogenized byultrasonic treatment. After homogenization, the supernatant wasrecovered by centrifugation to obtain a cell extract. Further, theEscherichia coli JM109 strain carrying plasmid pTrc101 was treated inthe same manner to prepare a cell extract.

The cell extracts thus prepared were measured for their enzymaticactivity. Namely, 50 μl of the cell extract, 200 μl of distilled water,and 250 μl of a substrate solution [2 mg/ml 4-amino-4-deoxychorismicacid, 10 mM magnesium chloride, 100 mM MOPS (Wako Pure ChemicalIndustries, Ltd.), pH 7.5] were mixed and reacted at 30° C. for 1 hour.After reaction, a portion of the reaction solution was analyzed using afull automatic amino acid analyzer JLC-500/V (JEOL, Ltd.).

As shown in FIG. 5, when the cell extract prepared from the Escherichiacoli carrying pTrc-papB was used, the peak for 4-amino-4-deoxychorismicacid declined and the peak for 4-amino-4-deoxyprephenic acid was newlydetected. A similar result was obtained when the cell extract boiled for5 minutes was used.

On the other hand, when the cell extract prepared from the Escherichiacoli carrying pTrc101 was used, there was no change in the peak for4-amino-4-deoxychorismic acid, and the peak for 4-amino-4-deoxyprephenicacid was not detected. Thus, these results revealed that the papB geneencodes 4-amino-4-deoxychorismic acid mutase and that the4-amino-4-deoxychorismic acid mutase encoded by the papB gene hadheat-resistant activity which was not lost even after boiling for 5minutes.

Example 4 Expression of papC Gene in Escherichia coli

In order to obtain the translation region of the papC gene, PCR wascarried out using the phage DNA derived from the positive clone shown inExample 1 as a template and oligonucleotides of SEQ ID NO: 13 and SEQ IDNO: 14 as primers. The PCR was carried out with KOD Dash (Toyobo Co.,Ltd.) as DNA polymerase using Gene Amp PCR System 9700 (Perkin-Elmer). Areaction solution containing 1 μl of phage DNA (equivalent to 1 μg), 5μl of 10-fold concentrated reaction buffer attached to the enzyme, 5 μlof a 2 mM dNTP solution, 1 μl each of the above-mentioned primersprepared at a concentration of 100 pmol/μl, 5 μl of dimethyl sulfoxide(Wako Pure Chemical Industries, Ltd.), 1 μl of KOD Dash and 31 μl ofsterile water was made up into a total volume of 50 μl. The reaction wascarried out by repeating incubation of 15 cycles of 30 seconds at 94°C., 2 seconds at 50° C. and 30 seconds at 72° C., after pretreatment at94° C. for 5 minutes. The reaction solution thus obtained was extractedwith phenol:chloroform:isoamyl alcohol (25:24:1) and precipitated withethanol. The precipitate was redissolved in sterile water, and digestedwith restriction enzyme BamHI, after which agarose gel electrophoresiswas carried out, and a DNA fragment of approximately 1 kbp was isolatedaccording to an ordinary method to recover a DNA fragment.

Plasmid pET-11c (Stratagene) was digested with restriction enzyme BamHIand treated with alkaline phosphatase (Takara Shuzo Co., Ltd.), afterwhich the resultant fragment was ligated to the above-mentioned 1 kbpBamHI DNA fragment using T4 DNA ligase. A plasmid into which the papCgene was inserted in the correct orientation to the promoter containedin pET-11c was selected and named pET-papC.

The nucleotide sequence of the inserted fragment of pET-papC wasdetermined using a fluorescent DNA sequencer ABI PRISM 310 GeneticAnalyzer (Perkin-Elmer) to verify that the sequence was identical withthe nucleotide sequence of SEQ ID NO: 5.

On the other hand, when the papC gene was expressed using pET-papC,evaluation of properties of papC gene products was expected to bedifficult since the vector-derived peptide composed of 14 amino acidswas added to the N-terminal side of the papC gene products. Therefore,pET-papC was digested with restriction enzyme NdeI, after which plasmidpET-papC1 was obtained by self-ligation using T4 DNA ligase. Use ofpET-papC1 made it possible to produce papC gene products by themselvesand not as fusion proteins. The above-mentioned plasmid constructionprocess is shown in FIG. 6.

The Escherichia coli BL21 (DE3) strain carrying pET-papC1 was culturedin an LB liquid medium (1% Bacto-tryptone, 0.5% yeast extract, 0.5%sodium chloride) supplemented with 100 μg/ml ampicillin, at 37° C.overnight. A 1 ml portion of the resultant culture was inoculated into100 ml of the same medium, and incubation was carried out at 37° C. for2 hours, after which 1 ml of 100 mM isopropylthiogalactoside (IPTG) wasadded, and incubation was further carried out at 37° C. for 5 hours.After incubation, cells were recovered by centrifugation, suspended in 4ml of buffer solution for cell homogenization (50 mM Tris-HCl (pH 8.0),5 mM EDTA, 10% glycerol), and then homogenized by ultrasonic treatment.After homogenization, the supernatant was recovered by centrifugation toobtain a cell extract. Further, the Escherichia coli BL21 (DE3) straincarrying plasmid pET-11c was treated in the same manner to prepare acell extract.

The cell extracts thus prepared were measured for their enzymaticactivity. Namely, 40 μl of the cell extract, 10 μl of the cell extractwhich was prepared from the Escherichia coli carrying pTrc-papBdescribed in Example 3 and boiled, 190 μl of distilled water, 10 μl of a10 mM NAD solution, and 250 μl of a substrate solution [2 mg/ml4-amino-4-deoxychorismic acid, 10 mM magnesium chloride, 100 mM MOPS(Wako Pure Chemical Industries, Ltd.), pH 7.5] were mixed and reacted at30° C. for 1 hour. After reaction, a portion of the reaction solutionwas analyzed using a full automatic amino acid analyzer JLC-500/V (JEOL,Ltd.).

As shown in FIG. 7, when the cell extract prepared from the Escherichiacoli carrying pET-papC1 was used, the peak for 4-amino-4-deoxychorismicacid declined, and the peak for 4-amino-4-deoxyprephenic acid to begenerated by the papB gene products disappeared. Sincep-aminophenylpyruvic acid cannot be detected by the full automatic aminoacid analyzer JLC-500/V, its synthesis could not directly be confirmed.

However, a peak for p-aminophenylalanine was detected. This wasgenerated probably due to the transfer of an amino group ofp-aminophenylpyruvic acid generated from papC gene products, byEscherichia coli aminotransferase. On the other hand, when the cellextract boiled and the cell extract which was prepared from theEscherichia coli carrying pET-11c were used, there was no change in thepeak for 4-amino-4-deoxyprephenic acid generated from papB geneproducts. Thus, it was revealed that the papC gene coded for4-amino-4-deoxyprephenic acid dehydrogenase.

Example 5 Construction of Plasmids pPF260-A2 and pPF260-A3 forIntroduction into PF1022 Producing-Microorganism

Plasmids pPF260-A2 and pPF260-A3 for expressing the papA gene in aPF1022-producing microorganism were constructed as shown in FIG. 8.

An expression vector pABPd for a PF1022-producing microorganism wasconstructed, and then the DNA fragment obtained from plasmid pUC118-papAdescribed in Example 2 was ligated to this vector to obtain anexpression vector. More specifically, the expression vector wasconstructed as described below.

Isolation of Genomic DNA of Substance PF1022-Producing Microorganism

The genomic DNA of the strain PF1022-producing strain (FERM BP-2671) wasisolated according to the method of Horiuchi et al. (H. Horiuchi et al.,J. Bacteriol., 170, 272–278, 1988). More specifically, cells of thesubstance PF1022-producing strain (FERM BP-2671) were cultured for 2days in a seed medium (2.0% soluble starch, 1.0% glucose, 0.5%polypeptone, 0.6% wheat germ, 0.3% yeast extract, 0.2% soybean cake, and0.2% calcium carbonate; pH 7.0 before sterilization; see Example 1 in WO97/00944), and the cells were recovered by centrifugation (3500 rpm, 10minutes). The cells thus obtained were lyophilized, suspended in a TEsolution, treated in a 3% SDS solution at 60° C. for 30 minutes, andthen subjected to TE-saturated phenol extraction to remove the celldebris. The extract was precipitated with ethanol and treated withRibonuclease A (Sigma) and Proteinase K (Wako Pure Chemical Industries,Ltd.), and then the nucleic acid was precipitated with 12% polyethyleneglycol 6000. The precipitate was subjected to TE-saturated phenolextraction and ethanol precipitation, and the resulting precipitate wasdissolved in a TE solution to obtain the genomic DNA.

Construction of Genome Library of Substance PF1022-ProducingMicroorganism

The genomic DNA derived from the substance PF1022-producingmicroorganism prepared as described above was partially digested withSau3AI. The product was ligated to the BamHI arm of a phage vector,λEMBL3 Cloning Kit (Stratagene) using T4 ligase (Ligation Kit Ver. 2;Takara Shuzo Co., Ltd.). After ethanol precipitation, the precipitatewas dissolved in a TE solution. The entire ligated mixture was used toinfect Escherichia coli LE392 strain using a Gigapack III Plus PackagingKit (Stratagene) to form phage plaques. The 1.3×10⁴ (2.6×10⁴ PFU/ml)phage library obtained by this method was used for cloning of the Abp1gene.

Cloning of the Abp1 Gene from the Genomic DNA Derived from SubstancePF1022-Producing Microorganism

A probe to be used was prepared by amplifying the translation region ofthe Abp1 gene by the PCR method. The PCR was carried out using thegenomic DNA prepared from the substance PF1022-producing microorganismas described above as a template and synthetic primers 8-73U and 8-73R,according to a LETS GO PCR kit (SAWADY Technology). The PCR reaction foramplification was conducted by repeating 25 cycles of 30 seconds at 94°C., 30 seconds at 50° C., and 90 seconds at 72° C. DNA sequences of the8-73U and 8-73R are as follows:

8-73U: CTCAAACCAGGAACTCTTTC (SEQ ID NO: 15)

8-73R: GACATGTGGAAACCACATTTTG (SEQ ID NO: 16)

The PCR product thus obtained was labeled using an ECL Direct System(Amersham Pharmacia Biotech). The phage plaque prepared as describedabove was transferred to a Hybond N+ nylon transfer membrane (AmershamPharmacia Biotech), and after alkaline denaturation, the membrane waswashed with 5×SSC(SSC: 15 mM trisodium citrate, 150 mM sodium chloride)and dried to immobilize the DNA. According to the kit protocol,prehybridization (42° C.) was carried out for 1 hour, after which theabove-mentioned labeled probe was added, and hybridization was carriedout at 42° C. for 16 hours. The nylon membrane was washed according tothe kit protocol described above. The washed nylon membrane was immersedfor one minute in a detection solution and then photosensitized on amedical X-ray film (Fuji Photo Film Co., Ltd.) to obtain one positiveclone. Southern blot analysis of this clone showed that a HindIIIfragment of at least 6 kb was identical with the restriction enzymefragment long of the genomic DNA. FIG. 9 shows the restriction map ofthis HindIII fragment. The HindIII fragment was subcloned into pUC119 toobtain pRQHin/119 for use of the following experiment.

Construction of Expression Vector

The promoter region and the terminator region of the Abp1 gene wereamplified by the PCR method using pRQHin/119 as a template. The PCRmethod was carried out using a PCR Super Mix High Fidelity (LifetechOriental Co., Ltd.) with primers ABP-Neco and ABP-Nbam for promoteramplification and ABP-Cbam and ABP-Cxba for terminator amplification.The amplification reaction was conducted by repeating 25 cycles of 30seconds at 94° C., 30 seconds at 50° C. and 90 seconds at 72° C. The DNAsequences of ABP-Neco, ABP-Nbam, ABP-Cbam and ABP-Cxba are as follows:

ABP-Neco: GGGGAATTCGTGGGTGGTGATATCATGGC (SEQ ID NO: 17)

ABP-Nbam: GGGGGATCCTTGATGGGTTTTGGG (SEQ ID NO: 18)

ABP-Cbam: GGGGGATCCTAAACTCCCATCTATAGC (SEQ ID NO: 19)

ABP-Cxba: GGGTCTAGACGACTCATTGCAGTGAGTGG (SEQ ID NO: 20)

Each PCR product was purified with a Microspin S-400 column (AmershamPharmacia Biotech) and precipitated with ethanol, after which thepromoter was double-digested with EcoRI and BamHI, the terminator wasdouble-digested with BamHI and XbaI, and the resulting fragments wereligated one by one to pBluescript II KS+ previously digested with thesame enzymes. The product was digested with XbaI, and a destomycinresistance cassette derived from pMKD01 (WO 98/03667) was inserted toconstruct pABPd (FIG. 10). pABPd has the promoter and terminator of theAbpI gene.

An approximately 2 kbp BclI DNA fragment was prepared from plasmidpUC118-papA described in Example 2. This fragment was inserted into theBamHI site of the expression vector pABPd for PF1022-producingmicroorganism to obtain plasmid pPF260-A.

Next, pPF260-A was double-digested with restriction enzymes PstI andBamHI to prepare a DNA fragment of approximately 1.7 kbp. This fragmentwas subcloned into PstI and BamHI sites of pUC119 to obtain plasmidpUC119-A. Treatment for site-directed mutagenesis was carried out withpUC119-A as a template DNA and the oligonucleotide of SEQ ID NO: 21 as aprimer using a Muta-Gene in vitro Mutagenesis Kit (Bio-Rad) to obtainplasmid pUC119-A1.

Next, pUC119-A1 and pPF260-A were double-digested with restrictionenzymes PstI and BamHI to prepare DNA fragments of approximately 1.7 kbpand approximately 8.6 kbp, and then these fragments were ligated toobtain plasmid pPF260-A2. Further, pPF260-A2 was digested withrestriction enzyme XbaI and then self-ligated using T4 DNA ligase toobtain plasmid pPF260-A3.

Example 6 Construction of Plasmid pPF260-B3 for Introduction intoPF1022-Producing Microorganism

Plasmid pPF260-B3 for expressing the papB gene in a PF1022-producingmicroorganism was constructed as shown in FIG. 11.

An approximately 0.3 kbp BamHI DNA fragment was prepared from plasmidpTrc-papB described in Example 3. This fragment was inserted into theBamHI site of the expression vector pABPd (Example 5) to obtain plasmidpPF260-B. pPF260-B was digested with restriction enzyme XbaI and thenself-ligated using T4 DNA ligase to obtain plasmid pPF260-B1.

Next, pPF260-B1 was digested with restriction enzyme PstI to prepare aDNA fragment of approximately 0.6 kbp. This fragment was subcloned intothe PstI site of pUC118 in such a manner that the papB gene and thelacZ′ gene aligned in the same direction to obtain plasmid pUC118-B.Treatment for site-directed mutagenesis was carried out with pUC118-B asa template DNA and the oligonucleotide of SEQ ID NO: 22 as a primerusing a Muta-Gene in vitro Mutagenesis Kit (Bio-Rad) to obtain plasmidpUC118-B1.

Next, pUC118-B1 and pPF260-B1 were digested with restriction enzyme PstIto prepare DNA fragments of approximately 0.6 kbp and approximately 8.0kbp, and then these fragments were ligated to obtain plasmid pPF260-B3.

Example 7 Construction of Plasmid pPF260-C3 for Introduction intoPF1022-Producing Microorganism

Plasmid pPF260-C3 for expressing the papC gene in a PF1022-producingmicroorganism was constructed as shown in FIG. 12.

An approximately 1 kbp BamHI DNA fragment was prepared from plasmidpET-papC described in Example 4. This fragment was inserted into theBamHI site of the expression vector pABPd (Example 5) to obtain plasmidpPF260-C pPF260-C was digested with restriction enzyme XbaI and thenself-ligated using T4 DNA ligase to obtain plasmid pPF260-C1.

Next, pPF260-C1 was double-digested with restriction enzymes PstI andSphI to prepare a DNA fragment of approximately 1.7 kbp. This fragmentwas subcloned into the PstI and SphI sites of pUC118 to obtain plasmidpUC118-C. Treatment for site-directed mutagenesis was carried out withpUC118-C as a template DNA and the oligonucleotide of SEQ ID NO: 23 as aprimer using a Muta-Gene in vitro mutagenesis kit (Bio-Rad) to obtainplasmid pUC118-C1.

Next, pUC118-C1 and pPF260-C1 were double-digested with restrictionenzymes PstI and SphI to prepare DNA fragments of approximately 1.7 kbpand approximately 7.6 kbp, and then these fragments were ligated usingT4 DNA ligase to obtain plasmid pPF260-C3.

Example 8 Transformation of PF1022-Producing Microorganism

A mixture of 1 μl of pPF260-A2, 3 μl of pPF260-A3, 3 μl of pPF260-B3,and 3 μg of pPF260-C3 was precipitated with ethanol and then redissolvedin 10 μl of TE buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA). The DNAsolution thus prepared was used to transform a PF1022-producingmicroorganism according to the method described in Example 1 of WO97/00944. More specifically, the PF1022-producing microorganism wascultured in the seed medium described in Example 5 at 26° C. for 48hours. After cultivation, the resultant mycelia were collected bycentrifugation at 3000 rpm for 10 minutes and washed with a 0.5 Msucrose solution. The mycelia thus obtained were subjected to protoplastgeneration by shaking in a 0.5M sucrose solution containing 3 mg/mlβ-glucuronidase (Sigma), 1 mg/ml chitinase (Sigma) and 1 mg/ml zymolyase(Seikagaku Kogyo) at 30° C. for 2 hours. The mixture thus obtained wasfiltered to remove the cell debris. The protoplasts were washed twice bycentrifugation (2500 rpm, 10 minutes, 4° C.) in an SUTC buffer solution(0.5M sucrose, 10 mM Tris-HCl (pH 7.5), 10 mM calcium chloride), andthen a 1×10⁷/ml protoplast suspension was prepared with the SUTC buffersolution.

The previously prepared plasmid DNA solution was added to 100 μl of theprotoplast suspension, and the resultant mixture was allowed to standunder ice-cooling for 5 minutes. Then, 400 μl of a polyethylene glycolsolution [60% polyethylene glycol 4000 (Wako Pure Chemical Industries,Ltd.), 10 mM Tris-HCl (pH 7.5), 10 mM calcium chloride] was added tothis mixture, and the resultant admixture was allowed to stand underice-cooling for 20 minutes.

The protoplasts treated as described above were washed with the SUTCbuffer solution and resuspended in the same buffer solution. Theresultant suspension was double-layered together with a potato dextrosesoft agar medium onto a potato dextrose agar medium containing 100 μg/mlhygromycin B and 0.5M sucrose. Incubation was carried out at 26° C. for5 days, and colonies appeared were deemed to be transformants.

Chromosomal DNAs were obtained from the resultant transformants, and PCRwas carried out using them as a temperate DNA under the same conditionsdescribed in Examples 2, 3 and 4, except that 25 cycles were repeated,to detect the papA, papB and papC genes. As a result, the 55-65 strain(FERM BP-7255) was selected as a transformant into which all of thethree genes were introduced.

Example 9 Cultivation of Transformed PF1022-Porducing Microorganism andDetection of PF1022 Derivative

The transformant strain 55-65 (FERM BP-7255) selected in Example 8 andthe parent strain were cultured as described in WO 97/20945. Namely,cells were cultured in the seed medium described in Example 5 at 26° C.for 2 days. A 2 ml portion of each resultant culture was inoculated into50 ml of a production medium (0.6% wheat germ, 1.0% pharma media, 2.6%soluble starch, 6.0% starch syrup, 0.2% MgSO₄ 7H₂O, 0.2% NaCl), andincubation was further carried out at 26° C. for 6 days. Afterincubation, the resulting cells were collected from a 40 ml portion ofthe culture by centrifugation and then extracted with 30 ml of ethylacetate. The extract was concentrated by drying and redissolved in 2 mlof acetonitrile. A 10 μl portion of the solution was subjected to HPLCanalysis.

Conditions for HPLC analysis were as follows:

HPLC system—655A-11, Hitachi, Ltd.

Column—Inertsil ODS-2, 4.6×250 mm

Mobile phase—Acetonitorile:water=70:30

Flow rate—1.0 ml/min

Column temperature—40° C.

Detector—870-UV, Nihon Bunko K. K.

UV wave length—245 nm

As shown in FIG. 13, the extract from the transformant strain 55-65exhibited the peaks each showing the same retention time with PF1022-268(cyclo[MeLeu-Lac-MeLeu-(O₂N)PhLac-MeLeu-Lac-MeLeu-PhLac]; Example 1 inWO 97/11064) and PF1022-269(cyclo[MeLeu-Lac-MeLeu-(H₂N)PhLac-MeLeu-Lac-MeLeu-PhLac]; Example 2 inWO 97/11064). On the other hand, neither of these peaks was detected forthe parent strain. Further, HPLC analysis using a mixture of the extractderived from the transformant and each standard verified that the peaksderived from the extract and the standard perfectly matched.Measurements of mass spectra using LC-MS (a quadrapole-type bench topLC/MS system NAVIGATOR with aQa™, Thermoquest) for the substancescontained in these peaks agreed with those for the standards.

From the results above, it was revealed that the transformant 55-65strain into which all of the three genes, i.e., the papA, papB and papCgenes, were introduced produced the substance PF1022 derivatives inwhich a benzene ring is modified at the para-position with a nitro groupor amino group.

1. A transformant of a microorganism, wherein the transformant isproduced by introducing (i) a polynucleotide encoding the amino acidsequence of SEQ ID NO: 2, (ii) a polynucleotide encoding the amino acidsequence of SEQ ID NO: 4, and (iii) a polynucleotide encoding the aminoacid sequence of SEQ ID NO: 6, into the microorganism, wherein themicroorganism to be transformed produces a peptide or a depsipeptide,which is substance PF1022 ([cyclo(D-lactyl-L-N-methylleucyl-D-3-phenyllactyl-L-N-methylleucyl-D-lactyl-L-N-methylleucyl-D-3-phenyllactyl-L-N-methylleucyl)])represented by the following formula:

and wherein the transformant produces a derivative of substance PF1022represented by the following formula:


2. The transformant according to claim 1, wherein the peptide or thedepsipeptide is synthesized from at least one molecule selected from thegroup consisting of phenylalanine, tyrosine, and phenyllactic acid. 3.The transformant according to claim 1, wherein the microorganism istransformed by introducing polynucleotides comprising: (i) the DNAsequence of SEQ ID NO: 1, (ii) the DNA sequence of SEQ ID NO: 3, and(iii) the DNA sequence of SEQ ID NO: 5 into the microorganism.
 4. Thetransformant according to claim 1, wherein the transformant is strain55-65 deposited with the National Institute of Bioscience andHuman-Technology under an accession number of FERM BP-7255.
 5. Thetransformant according to claim 1 wherein substance PF1022 issynthesized by a substance PF1022-synthesizing enzyme from fourmolecules of L-leucine, two molecules of D-lactic acid and two moleculesof D-phenyllactic acid.
 6. A method for producing a peptide or adepsipeptide having a benzene ring skeleton substituted at thepara-position with a nitro group or amino group, which comprises:culturing the transformant of claim 1 under conditions suitable forproduction of the peptide or the depsipeptide, and collecting thepeptide or the depsipeptide.
 7. A method for producing a substancePF1022 derivative, which comprises: culturing the transformant of claim1 under conditions suitable for production of the substance PF1022derivative, and collecting the substance PF1022 derivative.
 8. Anisolated polynucleotide encoding the amino acid sequence of SEQ ID NO:2.
 9. The polynucleotide according to claim 8, which comprises the DNAsequence of SEQ ID NO:
 1. 10. An isolated polynucleotide encoding theamino acid sequence of SEQ ID NO:
 4. 11. The polynucleotide according toclaim 10, which comprises the DNA sequence of SEQ ID NO:
 3. 12. Anisolated polynucleotide encoding the amino acid sequence of SEQ ID NO:6.
 13. The polynucleotide according to claim 12, which comprises the DNAsequence of SEQ ID NO:
 5. 14. A transformant of Mycelia sterilia,wherein the transformant is produced by transforming the Myceliasterilia by introducing (i) a polynucleotide encoding the amino acidsequence of SEQ ID NO: 2, (ii) a polynucleotide encoding the amino acidsequence of SEQ ID NO: 4, and (iii) a polynucleotide encoding the aminoacid sequence of SEQ ID NO:
 6. 15. The transformant according to claim14, wherein Mycelia sterilia is strain PF1022 deposited with theNational Institute of Bioscience and Human-Technology under an accessionnumber of FERM BP-2671.
 16. The transformant according to claim 14,wherein Mycelia sterilia is transformed by introducing polynucleotidescomprising (i) the DNA sequence of SEQ ID NO: 1, (ii) the DNA sequenceof SEQ ID NO: 3, and (iii) the DNA sequence of SEQ ID NO: into theMycelia sterilia.
 17. The transformant according to claim 14, whereinthe Mycelia sterilia to be transformed produces a substance PF1022([cyclo(D-lactyl-L-N-methylleucyl-D-3-phenyllactyl-L-N-methylleucyl-D-lactyl-L-N-methylleucyl-D-3-phenyllactyl-L-N-methylleucyl)]),represented by the following formula:


18. The transformant according to claim 17, wherein substance PF1022 issynthesized by a substance PF1022-synthesizing enzyme from fourmolecules of L-leucine, two molecules of D-lactic acid and two moleculesof D-phenyllactic acid.
 19. The transformant according to claim 14,wherein the derivative represented by the following formula: